CN108822210A - Pteromalus Puparum L venom Kazal-type serpin PpSPI24 albumen and application - Google Patents

Abstract

The invention discloses a Kazal-type serine protease inhibitor protein PpSPI24 of the chrysalis chrysalis venom, which has the amino acid sequence shown in SEQ ID NO:2. The present invention also discloses the gene encoding the above-mentioned Kazal-type serine protease inhibitor protein PpSPI24 of the wasp chrysalis venom at the same time, which has the nucleotide sequence at position 70-231 in SEQ ID NO:1. The inhibitors of the present invention can be used to inhibit the activation of PPO in the hemolymph of P. rapae or citrus swallowtail. The present invention also discloses a method for improving the preventive ability of cruciferous vegetables against lepidopteran pests, comprising transforming cruciferous vegetable cells with a gene having the nucleotide sequence shown in SEQ ID NO: 1, and then transforming the cruciferous vegetable cells into The resulting cruciferous vegetable cells are grown into plants.

Description

Chrysalis chrysalis venom Kazal-type serine protease inhibitor PpSPI24 egg white and application

technical field

The invention relates to the fields of molecular biology, genetic engineering and protein engineering. In particular, a Kazal-type serine protease inhibitor protein PpSPI24 expressed in the chrysalis chrysalis venom and its encoded nucleic acid sequence and application.

Background technique

Pests have always been a serious threat to the safe production of global crops, causing about a quarter of global food production to be reduced every year, resulting in huge economic losses. In my country, agricultural pests are also an important factor restricting the increase in agricultural production and the improvement of the quality of agricultural products. According to statistics, in 2014, the annual area of pests and diseases in the country was about 5.5 billion mu per time. The annual grain loss restored through the prevention and control of diseases and insect pests accounts for about 15%-20% of the total output. Even so, the annual grain loss can still reach 30 billion to 50 billion catties.

Since the advent of the chemical pesticide DDT, the control of pests has been mainly dependent on chemical pesticides. As a result, there have been problems such as rampant pests, insect resistance, pesticide poisoning, excessive pesticide residues, and serious pollution. People have been exploring to find new effective, safe and low-toxic pest control methods. With the emergence of transgenic technology, a new idea has been provided to solve the pest problem. Since the mid-1990s, the successful cultivation and application of insect-resistant transgenic crops has brought new opportunities for the effective control of pests. According to statistics, the global transgenic planting area has soared from about 1.7 million hectares in 1996 to 180 million hectares in 2014 (of which insect-resistant products accounted for 43%), which also produced obvious economic and ecological effects.

However, with the continuous planting of insect-resistant Bt crops with only a single Bt gene, the problem of target pest resistance to it has attracted increasing attention, and this concern has been reported on the American cotton bollworm Helicopera zea in Bt cotton fields. There are signs. For this reason, on the one hand, many scholars searched for and discovered new Bt insect-resistance genes, and also excavated protein/peptide genes with insecticidal activity from microorganisms, plants and animals (mainly scorpions and spiders). Or fusion genes and other means to breed new insect-resistant transgenic plants to delay or overcome the problem of pest resistance. As an important class of pest biological control agents, parasitoids have been fully affirmed in traditional biological control and play an important role in reducing the use of chemical pesticides and environmental pollution. Parasitic wasps can utilize a variety of parasitic factors carried by themselves (venom, polydnavirus (PDV), virus like particle (VLP), virus like filament (VLF), ovarian protein (ovarian protein, OP, teratocyte) to destroy host immune response, regulate host growth and development, regulate host hemolymph nutrient composition, disrupt host reproductive and endocrine system, etc. normal development. If the parasitic factors of parasitic wasps can be combined with modern biotechnology, it is expected to be used to develop new biocontrol agents or transgenic crops, which will open up a new way for pest biological control. For example, the secretory protein gene of the deformed cells of Microplitis croceipes has been successfully transferred into tobacco, and the growth of Manduca sexta has been significantly slowed down, and the degree of damage is significantly lower than that of the non-transgenic control, making tobacco It is insect resistant. Rodriguez-Andres et al. introduced the Egf1.0 protein in the parasitic factor PDV of Microplitis demolitor into Semliki Forest virus, which can significantly promote the proliferation of the virus and enhance its anti-Aedes aegypti The lethal effect of Aedes aegypti.

Contents of the invention

The technical problem to be solved by the present invention is to provide a Kazal-type serine protease inhibitor protein PpSPI24, which has an immunosuppressive effect on common agricultural pests (inhibits hemolymph melanization), and its encoded gene and application.

In order to solve the above technical problems, the present invention provides a serine protease inhibitor PpSPI24 from the chrysalis chrysalis venom, which has the amino acid sequence shown in SEQ ID NO:2.

Remarks: SEQ ID NO: 2 contains a signal peptide.

MAKSMLIVAFLLVACMMFMHVSA FTDQDGNNCPCATTDDLRYVCGSDGVTYDNESELNCENKCKRSNIQVRFEGKC.

As the improvement of the Kazal-type serine protease inhibitor protein PpSPI24 of the chrysalis golden wasp venom of the present invention: it is a protein, its conservative variant protein, its active fragment or its active derivative.

The present invention also simultaneously provides the gene encoding the above-mentioned chrysalis chrysalis venom Kazal-type 1 serine protease inhibitor protein PpSPI24, which has the nucleotide sequence of No. 70-231 in SEQ ID NO: 1; or with SEQ ID NO : The nucleotide sequence from nucleotide 70-231 in 1 has at least 70% homology; The nucleotide sequence of acid 70-231 is hybridized.

As an improvement of the gene of the present invention: the sequence of PpSPI24 contains 8-76 consecutive nucleotides (ie, 8-76 consecutive nucleotides in the nucleotide sequence at positions 70-231 in SEQ ID NO: 1).

The present invention also provides the purposes of the above-mentioned PpSPI24 of the chrysalis venom Kazal-type serine protease inhibitor protein: it is used to prepare the chrysalis chrysalis venom Kazal-type serine protease inhibitor protein, and the inhibitory protein can be used to inhibit Activation of PPO in the hemolymph of Cabbage butterfly.

The present invention also simultaneously provides a method for improving the preventive ability of cruciferous vegetables to lepidopteran pests, comprising transforming cruciferous vegetable cells with a gene having the nucleotide sequence shown in SEQ ID NO: 1, and then transforming the cruciferous vegetable cells into The resulting cruciferous vegetable cells are grown into plants.

The chrysalis golden wasp venom Kazal-type serine protease inhibitor protein PpSPI24 and its encoded nucleic acid sequence provided by the present invention make it possible to use its amino acid sequence, coding sequence and its development into valuable insect-resistant crops and Biopesticides are also used in many fields such as agricultural pest control.

In the present invention, the full-length sequence of the venom Kazal-type serine protease inhibitor protein PpSPI24 is obtained by sequencing the genome of the chrysalis chrysalis, and the target gene is obtained through gene synthesis. After obtaining the amino acid sequence of the Kazal-type serine protease inhibitor protein PpSPI24 from the chrysalis golden wasp venom, it was expressed in prokaryotes and purified under non-denaturing conditions. The activation of oxidase precursor (prophenoloxidase, PPO) prevents it from forming active phenoloxidase (phenoloxidase, PO), which has the function of inhibiting host humoral immunity.

The present invention is specifically achieved through the following technical solutions: the isolated DNA molecules in the present invention include: a nucleotide sequence encoding the Kazal-type serine protease inhibitor protein PpSPI24 having the chrysalis chrysalis venom, and the nuclear The nucleotide sequence has at least 70% homology with the nucleotide sequence from nucleotide 70-231 in SEQ ID NO: 1; or the nucleotide sequence can be compared with The nucleotide sequence from nucleotides 70 to 231 in SEQ ID NO: 1 hybridizes. Preferably, the sequence encodes a protein having the amino acid sequence shown in SEQ ID NO:2. More preferably, the said sequence has the nucleotide sequence from nucleotide 70-231 in SEQ ID NO:1.

The serine protease inhibitor protein PpSPI24 from chrysalis chrysalis venom isolated in the present invention includes: a protein having an amino acid sequence of SEQ ID NO: 2, or a conservative variant protein thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the protein is a protein having the sequence of SEQ ID NO:2.

The DNA molecule of the present invention comprises 8-76 consecutive nucleotides in the DNA molecule.

The host cells transformed with the DNA molecules of the present invention are prokaryotic cells.

In the present invention, "isolated", "purified" DNA refers to that the DNA or fragment has been separated from the sequences on both sides of it in the natural state, and also refers to that the DNA fragment has been combined with the accompanying nucleosides in the natural state. The components of the acid are separated and have been separated from the proteins that accompany it in the cell.

In the present invention, the nucleic acid sequence encoded by the chrysalis chrysalis venom Kazal-type serine protease inhibitor protein PpSPI24 refers to: the nucleotide encoding the protein having the activity of the chrysalis chrysalis chrysalis venom Kazal-type serine protease inhibitor protein PpSPI24 Sequence, such as the 70th-231st nucleotide sequence in SEQ ID NO: 1 and its degenerate sequence. The degenerate sequence refers to a sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the 70-231 nucleotides of the coding frame of SEQ ID NO: 1. Due to the degeneracy of the code, a degenerate sequence with as low as about 70% homology to the 70-231 nucleotide sequence in SEQ ID NO:1 can also encode the sequence described in SEQ ID NO:1.

Also included are nucleotide sequences capable of hybridizing to the nucleotide sequence from nucleotides 70 to 231 in SEQ ID NO: 1 under moderately stringent conditions, more preferably under high stringent conditions. Also includes at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology with the nucleotide sequence from nucleotide 70-231 in SEQ ID NO: 1 % of nucleotide sequences. Also included is a variant form of the open reading frame sequence in SEQ ID NO: 1 that can encode a protein that has the same function as the natural Kazal-type serine protease inhibitor protein PpSPI24 of the chrysalis chrysalis venom. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion/or substitution, and several additions at the 5' and/or 3' ends (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) Nucleotides.

In the present invention, the Kazal-type serine protease inhibitor PpSPI24 or protein of the chrysalis venom Kazal-type serine protease inhibitor PpSPI24 refers to the protein having the sequence of SEQ ID NO: 2 having the activity of the Kazal-type serine protease inhibitor PpSPI24 of the chrysalis chrysalis venom. The term also includes variants of the sequence of SEQ ID NO: 2 that have the same function as the natural chrysalis venom Kazal-type serine protease inhibitor PpSPI24. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids /or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the Kazal-type serine protease inhibitor PpSPI24 from the chrysalis golden wasp venom.

In the present invention, the Kazal-type serine protease inhibitor PpSPI24 conservative variant protein of the chrysalis golden wasp venom refers to: compared with the amino acid sequence of SEQ ID NO: 2, there are at most 10, preferably at most 8, more preferably Up to 5 amino acids with similar or similar properties are replaced to form proteins.

The present invention also includes the Kazal-type serine protease inhibitor PpSPI24 or protein analogs of the chrysalis golden wasp venom. The difference between these analogues and natural Kazal-type serine protease inhibitors may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins listed above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of proteins such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications during protein synthesis and processing or during further processing steps. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their proteolytic properties or to optimize their solubility properties.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids and the like. When producing the chrysalis chrysalis venom Kazal-type serine protease inhibitor PpSPI24 protein of the present invention, the coding sequence of the chrysalis chrysalis venom Kazal-type serine protease inhibitor PpSPI24 can be operably connected to the expression control sequence, thereby The expression vector of Kazal-type serine protease inhibitor PpSPI24 from the chrysalis chrysalis venom was formed.

As used herein, "operably linked to" refers to a situation where some portion of a linear DNA sequence is capable of affecting the activity of other portions of the same linear DNA sequence. For example, the signal peptide (secretion leader sequence) DNA is operably linked to the protein DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the protein; if the promoter controls the transcription of the sequence, then it is operably linked to the A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position where it can be translated. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention host cells are prokaryotic cells. Commonly used prokaryotic host cells are referred to as E. coli cells.

The expression of the gene product of the Kazal-type serine protease inhibitor PpSPI24 of the chrysalis venom Kazal-type serine protease inhibitor PpSPI24 can also be analyzed by Northern blot technique or fluorescent quantitative PCR, that is, the RNA transcript of the Kazal-type serine protease inhibitor PpSPI24 of the chrysalis venom Presence and quantity in cells.

In addition, the nucleic acid molecule that can be used as a probe in the present invention usually has 8-76 consecutive amino acids of the nucleotide coding sequence of the chrysalis venom Kazal-type serine protease inhibitor PpSPI24, preferably has 15 - 50 consecutive nucleotides. The probe can be used to detect whether there is a nucleic acid molecule encoding the Kazal-type serine protease inhibitor PpSPI24 of the chrysalis chrysalis venom in a sample.

The invention relates to a method for detecting whether there is a Kazal-type serine protease inhibitor PpSPI24 nucleotide sequence in a sample, which comprises the steps of hybridizing the sample with the above-mentioned probe, and then detecting whether the probe is combined. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence of the Kazal-type serine protease inhibitor PpSPI24 of the chrysalis golden wasp venom, and can be located on both sides of the coding sequence or in between. Primers are generally 15-50 nucleotides in length.

In addition, according to the nucleotide sequence and amino acid sequence of the venom Kazal-type serine protease inhibitor PpSPI24 of the present invention, the chrysalis can be screened on the basis of nucleic acid homology or expressed protein homology. Venom Kazal-type serine protease inhibitor PpSPI24 homologous gene or homologous protein.

The full-length nucleotide sequence of the Kazal-type serine protease inhibitor PpSPI24 of the chrysalis chrysalis venom of the present invention or its fragment can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify to obtain related sequences.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis. Utilizing the chrysalis chrysalis venom Kazal-type serine protease inhibitor PpSPI24 of the present invention, through various conventional screening methods, the substances that interact with the chrysalis chrysalis venom Kazal-type serine protease inhibitor PpSPI24 can be screened out, or receptors, etc.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. The invention has obvious inhibitory effect in the hemolymph phenol oxidase activation test of the important pest of cruciferous vegetables, Pieris rapae, and has obvious inhibitory effect on the humoral immunity of the cabbage butterfly. my country's agricultural pests are very harmful, and the negative impact of using chemical pesticides is very large. The Kazal-type serine protease inhibitor PpSPI24 of the present invention is just a new protein that has an immunosuppressive effect on agricultural pests. Therefore, it has great Great application value.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is the separation and purification diagram of PpSPI24 prokaryotic expression of the present invention;

Note: M is the standard protein, lane 1 is the uninduced whole protein of Escherichia coli BL21 containing pGEX-4T-2-PpSPI24 plasmid, lane 2 is the supernatant after induction of Escherichia coli BL21 containing pGEX-4T-2-PpSPI24 plasmid, lane 3 is The purified Kazal-type serine protease inhibitor PpSPI24 protein fused with the GST tag in the chrysalis venom, lane 4 is the purified GST tagged protein.

Fig. 2 is the inhibitory effect diagram of the prokaryotic expression of the present invention's chrysalis chrysalis venom Kazal-type serine protease inhibitor PpSPI24 gene expression on the activation of cabbage chrysalis hemolymph phenol oxidase;

NOTE: M.luteus/GST-PpSPI24 represents 0.5 μg of PpSPI24 protein added to M.luteus.

Negative control was TBS buffer with M.luteus added; 0.5 μg tagged protein GST was added with M.luteus; 0.5 μg BSA standard protein was added with M.luteus; positive control was TBS buffer without M.luteus.

Detailed ways

Below in conjunction with laboratory specific test data and in conjunction with specific examples, further elaborate the present invention. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example 1:

1. Gene synthesis of the Kazal-type serine protease inhibitor PpSPI24 from the chrysalis golden wasp venom:

According to the gene sequence obtained from the genome data of Chrysalis chrysalis ----- the sequence described in SEQ ID NO: 1, the signal peptide was predicted online by using SignalP 4.1. Gene sequence design and synthesis of single-stranded oligo, using PCR to splice the synthesized oligo into a complete gene sequence. Connected to the carrier PGEM-Teasy (Promega), screened by blue and white spots, picked positive clones, and sent them to Shanghai Boshang Company for sequencing inspection.

2. Prokaryotic expression and purification of PpSPI24

After obtaining the full length of PpSPI24, use the full-length ORF (remove the signal peptide) to design primers for constructing prokaryotic expression vectors:

Forward primer pGEX-4T-2-PpSPI24-SP:

5'-GATCTGGTTCCGCGTGGATCCTTTTACCGACCAGGACGGCA-3',

Reverse primer pGEX-4T-2-PpSPI24-AP:

5'-ACGATGCGGCCGCTCGAGTCAGCATTTGCCTTCGAACC-3'.

Using the gene fragment plasmid that has been connected to the carrier PGEM-T easy (Promega) as a template, PCR amplification was performed using the full gold high-fidelity Pfu enzyme. The PpSPI24 PCR amplification system was: 94°C for 3min, 94°C for 30sec, 51°C Anneal for 30 sec, extend for 30 sec at 72°C, and extend for 8 min at 72°C after 35 cycles. After the completion of PCR, it was verified by 1% agarose gel electrophoresis, and then the gel was cut and recovered for later use. Extract the pGEX-4T-2 plasmid, and double digest the pGEX-4T-2 plasmid with BamHI and XholⅠ;

The digestion steps are as follows:

1) Enzyme digestion reaction solution:

2) After centrifugation and mixing, react at 37°C for 2 hours, and then heat at 37°C for 20 minutes to inactivate the enzyme. After the digestion product is verified by agarose gel electrophoresis, it is recovered by gel cutting, and the linearized pGEX-4T-2 vector is obtained. Homologous recombination was performed using the ClonExpress Entry OneStep Cloning Kit. The reassembly steps are as follows:

Homologous recombination reaction solution:

3) Use a pipette to pipette up and down several times, gently mix the components, and place at 37°C for 30 minutes. Immediately after the completion of the reaction, the reaction tube was placed in an ice bath and cooled for 5 min. The recombinant plasmid was then transformed into E. coli BL21 Escherichia coli, and added to non-resistant LB liquid medium, cultured in a 37°C incubator at 200r for 1 hour, and then sucked 100 μL and inoculated it on an LB plate containing 100 μg/ml Amp+ for overnight culture. The next day, a single colony was picked and cultured in LB liquid medium containing Amp+ (the concentration of Amp+ was 100 μg/ml) and sent for sequencing to detect whether the expression vector was successfully constructed. Pick a single colony containing the expression plasmid (after extracting the single colony plasmid, sequence and verify that the plasmid contains the inserted PpSPI24 sequence, and the external performance is that it can grow on the LB solid medium containing Amp+) and inoculate it in 5ml LB medium (containing 100 μg/ ml Amp+), shake culture overnight at 37°C, take the above 100μl bacterial solution and culture it in fresh 5ml LB medium to OD 0.6-0.8 (about 2~3h), add 500mM IPTG 10μl to a final concentration of 0.5mM, and induce expression at 37°C After culturing for 4-5 hours, the cells were collected. The purification of the fusion PpSPI24 containing the GST tag (expressed in Escherichia coli using the pGEX-4T-2 expression vector) was performed according to the instructions of the Invitrogen ^™ GST Spin Purification Kit purification kit. The specific results of expression and purification are shown in FIG. 1 .

According to Fig. 1, it can be known that Escherichia coli BL21 containing the pGEX-4T-2-PpSPI24 plasmid was expressed in a large amount in the supernatant after induction, and the purified band was single, about 38 kDa.

3. Determination of the inhibitory effect of synthesized and expressed PpSPI24 on PPO activation in Cabbage butterfly and Citrus swallowtail

Take the hemolymph from the chrysalis chrysalis from one head on ice, put it in a 1.5ml pre-cooled centrifuge tube, put it on ice immediately, centrifuge at 3300g for 5 minutes at 4°C, and transfer the supernatant to a new pre-cooled 1.5ml centrifuge tube In the process, the blood cells were removed for the determination of the background of phenol oxidase.

The experimental loading is as follows:

Effects on PPO activation:

10 μl TBS (containing 2 μl pupal hemolymph),

10 μl TBS (containing 2 μl pupal hemolymph and 0.5 μg M.luteus),

10 μl TBS (containing 2 μl pupal hemolymph, 0.5 μg BSA and 0.5 μg M.luteus),

10 μl TBS (containing 2 μl pupal hemolymph, 0.5 μg GST and 0.5 μg M.luteus),

10 μl TBS (containing 2 μl pupal hemolymph, 1 μl PTU and 0.5 μg M.luteus),

10ul TBS (containing 2μl pupal hemolymph, 0.5μg GST-PpSPI expressed in GST fusion and 0.5μg M.luteus);

After standing at 25°C for 20 minutes, 200 μl of L-Dopa (20 mM, dissolved in PBS) was added, and detected once per minute under the absorbance value of 470 nm for a total of 30 minutes. Each sample was repeated at least 3 times. The phenoloxidase activity unit U refers to the value of 0.001 OD ₄₇₀ changed per minute. The data were analyzed by variance analysis using DPS data analysis software (Tang Qiyi and Feng Mingguang, 2007). The specific results are shown in Figure 2. The above test results prove that the PpSPI24 protein of the present invention has an inhibitory effect on the activation of PPO in P. rapae and citrus swallowtail.

Example 2

1. Construction of PpSPI24 gene plant binary expression vector

The cauliflower mosaic virus CaMV 35S promoter and NOS terminator on both sides of the GUS gene in the plant binary expression vector pBI121 were used to insert the PpSPI24 gene to form a complete expression framework. In this experiment, BamHI and SacⅠ were selected as restriction sites to replace the GUS gene with PpSPI24, so that the expression cassettes on both sides of the GUS gene were used to control the expression of the PpSPI24 gene in Arabidopsis plants.

Primers with BamHI and SacⅠ restriction sites were designed according to the ORF of the serine protease inhibitor of the venom of A. chrysalis, and the plant expression vector was constructed by PCR amplification using the cDNA of the venom gland of A. chrysalis as a template. The primer sequences are:

PpSPI24-SP:5'-ACGCGTCGACTTTACCGACCAGGACGGCA-3',

PpSPI24-AP:5'-CGCGGATCCTCAGCATTTGCCTTCGAACC-3',

Among them, GGATCC is the restriction site of BamHI, and GTCGAC is the restriction site of SacⅠ. Primers were synthesized by Shanghai Sangon Bioengineering Company.

The PCR reaction conditions and system were the same as in Example 1-2 (that is, the forward primer and reverse primer were replaced by "PpSPI24-SP and PpSPI24-AP" in the reaction system, respectively). The PCR product was recovered, ligated into the pMD18-T vector, transformed into Escherichia coli Trans T1 competent cells, screened for Amp+ resistance, and the clones were picked and sent to Shanghai Boshang Company for sequencing. The pMD18-PpSPI24 plasmid was extracted, and the pMD18-PpSPI24 plasmid was double digested with restriction endonucleases BamHI and SacⅠ, and small fragments were recovered by cutting the gel.

The pBI121 plasmid was extracted from the overnight cultured bacterial solution with a mini-plasmid extraction kit (Axygen), and the pBI121 plasmid was double-digested with restriction endonucleases BamHI and SacⅠ, and the large fragment was recovered by cutting the gel. The digested pBI121 plasmid and pMD18-PpSPI24 plasmid were ligated overnight at 16°C with T4 DNA ligase, transformed into Escherichia coli Trans T1 competent cells, screened for Kan+ resistance, and pBI121-PpSPI24 was picked (the sequence obtained by sequencing is shown in SEQ ID NO : 1) The clone was sent to Shanghai Boshang Company for sequencing to verify the correctness of the inserted fragment.

2. Transformation of Agrobacterium with pBI121-PpSPI24

(1) Preparation of Agrobacterium Competent Cells

a. Streak culture of EHA105 strain on YEP solid medium containing 50mg/L Rif, culture at 28°C for 24-48h;

b. Pick the monoclonal strain in 5ml of YEP liquid medium containing 50mg/L Rif, shake and culture at 28°C for 24-48h;

c. Add 5 ml of the above bacterial solution to 100 ml of YEP liquid medium containing 50 mg/L Rif, and culture with shaking at 28°C for 5-6 hours until OD ₆₀₀ =0.8.

d. Centrifuge at 5000 rpm for 15 min at 4°C to collect cell pellets. Add 50ml of ice-cold sterile water to suspend the bacterial pellet;

e. Centrifuge at 5000rpm for 15min at 4°C, then repeat the operation with 10ml of ice-cold sterile water;

f. Add 10ml of pre-cooled 10% sterile glycerol to resuspend the precipitate;

g. Centrifuge, discard the supernatant, suspend the precipitate with 2 ml of pre-cooled 10% sterile glycerol, aliquot and store at -70°C.

(2) Electric shock transformation of Agrobacterium

Take out the electric shock cup, first wash it twice with double distilled water, then wash it 1-2 times with 75% ethanol (after adding 75% ethanol, shake it twice), put it into the ultra-clean bench, and blow dry with the mouth facing inward (approximately 10-20 minutes), the electric shock cup after drying was covered with a lid and placed on ice.

Take a competent cell of Agrobacterium from a -70°C refrigerator, put it on ice, and when it melts into a liquid state, add 2ul pBI121-PpSPI24 plasmid to the competent cell in an ultra-clean bench, flick and mix well by hand Put it on ice, and then transfer it to the middle gap of the shock cup with a pipette. Turn on the electric shock instrument, dry the water outside the electric shock cup with absorbent paper, put in the electric shock cup, and rotate the gear until it is tight. Adjust the electric shock parameters (1440HV, 125Ω, 50uF), place on ice for 2-3 minutes after electric shock, then gently add 800ul LB medium to the electric shock cup, gently blow with a pipette a few times and then transfer to EP tube.

Culture at 28°C for 48 hours, centrifuge at 4000rpm for 5 minutes, and then coat the plates containing Kan+antibiotics. The grown colonies were first streaked on another plate, and after continuing to grow for 24 hours, PCR verification was performed. Because the copy number of the plasmid contained in Agrobacterium was very low, the number of PCR cycles should be set to 40 cycles.

3. Transformation of Arabidopsis thaliana with Agrobacterium

Arabidopsis transformation was performed using the pollen tube passage method (Clough and Bent 1998). The specific process is as follows:

Pick a single colony of Agrobacterium containing pBI121-PpSPI24 on the growing LB solid medium, culture it in 4ml LB liquid medium overnight, and expand the culture according to the ratio of 1:500. When the OD ₆₀₀ absorption value of the bacterial solution reaches 0.6-1.0, centrifuge at 4000 rpm for 5 minutes to collect the Agrobacterium.

Resuspend Agrobacterium in 5% sucrose solution, dilute the bacterial solution to OD ₆₀₀ of about 0.5-0.8, and add 0.03% surfactant Silvet L-77 before use. Select Arabidopsis thaliana in good growth state, soak the flower buds of Arabidopsis thaliana in the Agrobacterium liquid for 20 seconds. Shade and moisturize with opaque plastic cloth for 24 hours, and then transfer to light incubator to continue culturing. Repeat the conversion a week later.

The transformed Arabidopsis thaliana can be placed under long-day conditions, so that it grows rapidly and sets seeds, and the T ₀ generation seeds are collected.

4. Identification of transgenic Arabidopsis

(1) Kanamycin resistance screening of transgenic plants

a. Take about 100 mg of Arabidopsis _thaliana seeds harvested and put them into a 1.5ml centrifuge tube;

b. Disinfect with 70% alcohol for 5 seconds, centrifuge briefly and discard the supernatant;

c. Sterilize with 10% NaHClO ₃ for 2 to 3 minutes, and discard the supernatant after brief centrifugation;

d. Add sterilized distilled water to resuspend, discard the supernatant, and wash several times;

e. Add 1 ml sterile 0.1% agarose solution to suspend the seeds;

f, spreading Arabidopsis seeds on the 1/2MS medium containing 50mg/L Kan+;

g. Vernalization at 4°C for 2 to 5 days;

h, move to Arabidopsis thaliana under normal conditions for cultivation, after one week, the Arabidopsis seedlings resistant to Kan+ will appear, and the resistant seedlings will be transplanted into soil:frog stone=1:1 flower pot, put in the cultivation room Grow and collect T ₁ generation seeds.

(2) PCR identification of Arabidopsis

Take the young leaves of Arabidopsis thaliana seedlings _of the T1 generation, and extract the plant genomic DNA. The specific method is as follows:

a. Collect _one young leaf of the T1 generation into a 1.5ml centrifuge tube, inject liquid nitrogen, and grind the sample into powder;

b. Add 750 μl of extraction buffer to the centrifuge tube containing the sample, shake and mix quickly, and place the centrifuge tube at 65°C for 8-10 minutes;

c. Add 150μl 5M LiAc, mix gently, and ice bath for 15-20min;

d. Centrifuge at 13000rpm for 10min at 4°C;

e. Transfer 800 μl supernatant to a new centrifuge tube, add an equal volume of isopropanol, invert and mix well, and precipitate at -20°C for 10 minutes;

f. Centrifuge at 13000rpm for 10min at 4°C;

g, wash the precipitate with 75% ethanol, and dry;

h. Dissolve the precipitate in 10 μl TE buffer;

i. Dilute the extracted DNA sample 5 to 10 times as a PCR reaction template, perform PCR reaction according to the method of Example 1-1, and identify the transgene status of the T1 generation.

Seventy wild-type Arabidopsis plants were transformed with the Agrobacterium pollen tube method carrying the recombinant plasmid pBI121-PpSPI24, and thousands of seeds were harvested from each plant. Through the expression of the kana resistance gene, the seeds that have been introduced with the foreign gene were initially screened out, and about 35,000 seeds were sown, and finally 72 seeds with kana resistance were obtained. The plants were self-pollinated by wrapping in plastic bags to obtain T1 generation seeds, which _were then backcrossed until T3 generation homozygotes _were used for subsequent analysis.

5. Insect resistance analysis of PpSPI24 transgenic Arabidopsis plants

SEQ ID takes 10 2-year-old or so Pieris rapae and puts them in a 15cm petri dish, and places soaked cotton balls in the petri dish to keep moist. Add the same amount of rosette 8 leaf stage leaves of PpSPI24 Arabidopsis and wild-type Arabidopsis into each petri dish and allow them to eat, set 3 replicates for each treatment, and record the number of survival and dead amnesia in each treatment and The growth status of larvae was monitored, fresh leaves were replaced in time, and the mortality rate of Pieris rapae was counted. After feeding for 5 days, the mortality rate of Pieris rapae larvae fed on the leaves of PpSPI24-transformed Arabidopsis was 22%, which was significantly higher than the mortality rate of 4.2% of the control, indicating that the transgenic PpSPI24 Arabidopsis can inhibit the survival of Pieris rapae and is poisonous to Pieris rapae. effect. It is expected that the transfer of PpSPI24, the venom protein of the chrysalis chrysalis into other cruciferous vegetables such as cabbage, has a control effect on Lepidoptera pests such as cabbage caterpillar.

Finally, it should also be noted that the above examples are only specific implementation examples of the present invention. Apparently, the present invention is not limited to the above examples, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

sequence listing

<110> Zhejiang University

<120> Kazal-type serine protease inhibitor PpSPI24 protein from the chrysalis golden bee venom and its application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 231

<212>DNA

<213> Pteromalus puparum

<400> 1

atggccaagt ctatgctcat cgttgctttc ctcctcgtcg cctgcatgat gttcatgcac 60

gtgagcgcgt ttaccgacca ggacggcaac aactgtccct gtgccaccac tgacgatctc 120

agatatgttt gcggcagcga cggcgtcacc tacgacaacg agtctgagct caactgcgag 180

aacaagtgca aacgatccaa catccaggtt aggttcgaag gcaaatgctg a 231

<210> 2

<211> 76

<212> PRT

<213> Pteromalus puparum

<400> 2

Met Ala Lys Ser Met Leu Ile Val Ala Phe Leu Leu Val Ala Cys Met

1 5 10 15

Met Phe Met His Val Ser Ala Phe Thr Asp Gln Asp Gly Asn Asn Cys

20 25 30

Pro Cys Ala Thr Thr Asp Asp Leu Arg Tyr Val Cys Gly Ser Asp Gly

35 40 45

Val Thr Tyr Asp Asn Glu Ser Glu Leu Asn Cys Glu Asn Lys Cys Lys

50 55 60

Arg Ser Asn Ile Gln Val Arg Phe Glu Gly Lys Cys

65 70 75

Claims (6)

1. The Kazal-type serine protease inhibitor protein PpSPI24 of the chrysalis golden bee venom is characterized in that it has the amino acid sequence shown in SEQ ID NO:2. 2. The chrysalis chrysalis venom Kazal-type serine protease inhibitor protein PpSPI24 according to claim 1, characterized in that: said protein is protein, its conservative variant protein, its active fragment or its active derivative. 3. the gene encoding the chrysalis chrysalis venom Kazal-type serine protease inhibitor protein PpSPI24 as claimed in claim 1 or 2, is characterized in that: it has the nucleoside of No. 70-231 in SEQ ID NO:1 acid sequence; or have at least 70% homology with the nucleotide sequence from nucleotide 70-231 in SEQ ID NO: 1; or its nucleotide sequence can be compared with SEQ ID In NO:1, the nucleotide sequence from nucleotide 70-231 is hybridized. 4. The gene PpSPI24 according to claim 3, characterized in that: said sequence contains 8-76 consecutive nucleotides. 5. the purposes of the chrysalis chrysalis venom Kazal-type serine protease inhibitor protein PpSPI24 protein as claimed in claim 1, is characterized in that: be used for preparing chrysalis chrysalis chrysalis venom Kazal-type serine protease inhibitor protein PpSPI24 , the inhibitor can be used to inhibit the activation of PPO in the hemolymph of P. rapae or citrus swallowtail. 6. The method for improving the preventive power of cruciferous vegetables to lepidopteran pests is characterized in that: comprising transforming cruciferous vegetable cells with a gene having a nucleotide sequence shown in SEQ ID NO: 1, and then transforming the transformed cruciferous Flower vegetable cells are grown into plants.